ABSTRACT Cell-cell fusion promotes the development and physiology of most organs and has been documented in diseases including multiple types of cancer. Various reports have shown that stromal cells in the tumor microenvironment can fuse with neoplastic cells to generate malignant hybrid progeny. The pathophysiological significance of fusion events in cancer initiation and progression, however, remains largely uncharacterized. In this project, we will analyze how cell- cell fusion contributes to the pathogenesis of the malignant brain cancer astrocytoma. We hypothesize that astrocytoma growth and progression from low-grade status to high-grade glioblastoma (GBM) is driven, in part, by fusion between tumor cells and neural stem and progenitor cells (NPs) in the brain microenvironment. We base this hypothesis on the following unpublished experimental data presented in this application: (i) non-malignant NPs are recruited to early stage astrocytomas and readily incorporate into the tumor cytoarchitecture; (ii) NPs and astrocytoma cells can fuse to yield proliferative and poorly differentiated hybrid progeny with stem-like properties; (iii) tumor growth and progression to GBM correlate with NP recruitment and fusion with tumor cells; and (iv) human GBMs contain multinucleated cells that express NP markers that may arise via cell-cell fusion. To analyze mechanisms of pathological cell fusion in GBM we will generate and characterize a unique set of in vitro and in vivo systems. First, genetic-based lineage tracing experiments will be used to purify cell fusion hybrids and study self-renewal, multipotency, cytogenetic abnormalities and gene expression profiles. Second, functional roles for cell fusion hybrids in tumor growth and progression will be studied using transgenic pre-clinical mouse models of GBM. We will use genetic strategies to inhibit cell-cell fusion or kill newly generated fusion hybrids in vivo and quantify astrocytoma growth, invasion and progression to GBM. In summary, the experiments proposed in this project will reveal novel roles for resident NPs in the brain microenvironment in driving tumor progression and molecular genetic heterogeneity via cell-cell fusion.